PT643608E - PURIFICATION OF PROTEINS - Google Patents

Abstract

This invention relates to the application of combination chromatography to the purification of complement receptor proteins.

Description

84 966 ΕΡ Ο 643 608 / ΡΤ

"Protein purification"

Field of the invention

This invention relates to the field of protein purification. More specifically, this invention relates to the application of chromatography in combination for the purification of proteins from the receptor to the complement. In particular, this invention relates to the application of various combinations of ion exchange chromatography, optional precipitation, HIC and size exclusion, for the purification of molecules similar to those of the receptor for the complement.

BACKGROUND OF THE INVENTION

Historically, schemes for purifying proteins have been based on differences in the molecular properties of size, charge and solubility between the protein to be purified and unwanted protein contaminants. Protocols based on these parameters include size exclusion chromatography, ion exchange chromatography, differential precipitation and the like. Size exclusion chromatography, also known as gel filtration or gel permeation chromatography, relies on the penetration of macromolecules in a mobile phase into the pores of stationary phase particles. Differential penetration is a function of the hydrodynamic volume of the particles. Accordingly, under ideal conditions the larger molecules are excluded from the interior of the particles while the smaller molecules have access to this volume and the order of evasion can be predicted on the basis of the size of the protein given there is a linear relationship between the volume of evasion and the logarithm of molecular weight. Chromatographic supports for size exclusion based on cross-linked dextrans, eg SEPHADEX®, on spherical agarose beads, eg SEPHAROSE® (both commercially available from Pharmacia AB, Uppsala, Sweden), based on crosslinked polyacrylamides , eg BIO-GEL® (commercially available from BioRad Laboratories, Richmond, California), or based on ethylene glycol-methacrylate copolymer, eg TOYOPEARL HW65S (commercially available from ToyoSoda Co., Tokyo, Japan).

84 966 ΕΡ Ο 643 608 / ΡΤ 2

Precipitation methods are based on the fact that, in blends of crude proteins, it is likely that the solubilities of the individual proteins will vary widely. Although the solubility of a given protein in an aqueous medium depends on a variety of factors, it is generally stated in the discussion that a protein will be soluble if its interaction with the solvent is stronger than its interaction with protein molecules of the same or similar type. Without wishing to be bound by any particular mechanistic theory describing the phenomenon of precipitation, however, it is found that the interaction between a protein and the water molecules occurs by means of hydrogen bonds with various types of groups without charge and electrostatically as dipoles, with charged groups, and that precipitants such as monovalent cation salts (eg ammonium sulfate) compete with the proteins for the water molecules, thus at high salt concentrations, the proteins become "dehydrated" by reducing their interaction with the medium aqueous and increasing aggregation with the same or similar proteins resulting in precipitation from the medium. Ion exchange chromatography involves the interaction of functional groups with charge in the sample with ionic functional groups of opposite charge on an adsorbent surface. Two general types of interaction are known. Negatively charged amino acid (eg, aspartic acid and glutamic acid) side-chain-mediated anion exchange chromatography interacting with positively charged surfaces, and positively charged amino acid residue-mediated cation exchange chromatography (eg lysine and arginine) interacting with charged surfaces negatively.

More recently, affinity chromatography and hydrophobic interaction chromatography techniques have been developed to supplement the more traditional size exclusion and ion exchange chromatographic protocols. Affinity chromatography relies on the interaction of the protein with an immobilized ligand. The ligand may be specific for the protein of particular interest, in which case the ligand is a substrate, substrate analogue, inhibitor or antibody. Altematively, the ligand may be able to react with a number of proteins. Said general ligands, such as adenosine monophosphate, adenosine diphosphate, nicotine adenine dinucleotide or certain dyes may be employed in order to recover a particular class of proteins.

84 966 ΕΡ Ο 643 608 ΡΤ Hydrophobic interaction chromatography was first developed after observing that proteins may be retained on affinity gels which comprise hydrocarbon spacer arms but lack the affinity ligand. Although in this field the term hydrophobic chromatography is sometimes used, the term hydrophobic interaction chromatography (HIC) is preferred since it is the interaction between the solute and the gel which is hydrophobic, not the chromatographic process. The hydrophobic interactions are stronger under high ionic strength, so this separation form is conveniently performed after saline or ion exchange processes. The evasion from supports for HIC may be carried out by changes in solvent, pH, ionic strength, or by the addition of chaotropic agents or organic modifiers, such as ethylene glycol. A description of the general principles of hydrophobic interaction chromatography can be found in US-A-3 917 527 and US-A-4 000 098. The application of HIC to the purification of specific proteins is exemplified by reference to the following descriptions: growth hormone (US-A-4 332 717), toxin conjugates (US-A-4,771,128), anti-hemolytic factor (US-A-4,743,680), tumor necrosis factor (US-A-4,894,439 ), interleukin-2 (US-A-4,908,434), human lymphotoxin (US-A-4,920,196) and lysozyme species (Fausnaugh, JL and FE Regnier, J. Chromatog., 359: 131-146 (1986) )).

Yeh, C. G. et al., The Journal of Immunology 146: 250-256 (1991) describe the purification of culture supernatant from sCR1 containing cells using cation exchange chromatography. The invention provides a method as defined in the present claim 1 with some preferred embodiments being the object of the dependent claims, the embodiments of claims 5 and 8 being particularly preferred.

Detailed Description of the Invention

This invention relates to techniques for the purification of proteins, which are useful in the large-scale purification of proteins from the receptor to the complement. The invention is particularly useful in that it allows the recovery of protein from the receptor with protein purity> 95%. The invention may be applied to the purification of a number of proteins from the receptor to the complement and from proteins similar to those of the receptor to the complement.

84 966 ΕΡ Ο 643 608 / ΡΤ Ο complement is a group of serum proteins, sequentially activated by limited proteolysis, which are important effectors of humoral immunity. Complement activation occurs by the interaction of early acting complement components with antigen / antibody complexes. The proteolytic fragments resulting from this activation, alone or with other proteins, activate additional complement proteins, yielding a proteolytic cascade that recalls the functioning of blood coagulation factors. Altematively, the complement may be activated by components of the bacterial cell wall, proteolytic enzymes (e.g. plasmin) or complex carbohydrates (e.g., inulin). A number of biological activities are mediated by components of the complement system (e.g., immune cytolysis, anaphylactic toxin production, bacteriolysis, chemotaxis, hemolysis, opsonization, and phagocytosis). Four classes of complement receptors (CR) (CR1-CR4) are known. The complement receptor 1 (CRI) is a receptor for complement components C3b and C4b. The complement receptor 2 (CR2) is a receptor for the C3dg or C3d component. The receptor for complement 3 (CR3) is a receptor for C3bi. The complement receptor 4 (CR4) is a receptor for the C3dg component. The complement receptor type 1 (CRI) is present in the membranes of erythrocytes, monocytes / macrophages, granulocytes, B cells, some T cells, splenic follicular dendritic cells, and glomerular podocytes. The CRI binds C3b and C4b and is termed the C3b / C4b receptor. Its primary sequence was determined (Klickstein et al., J. Exp. Med. 165: 1095-1112 (1987), Klickstein et al., J. Med., 168: 1699-1717 (1988), Hourcade et al. al. J. Med. 168: 1255-1270 (1988)). It consists of 30 short consensual replicates (SCRs) containing 60-70 amino acids, of which 29 are preserved from the mean of 65 amino acids per SCR. It is proposed that each SCR forms a triple-loop three-dimensional structure through disulfide bonds with the third and the first and the fourth and the second half-cystines on disulfide bonds. SCRs are further organized into 4 long homologous repeats (LHR) of 7 SCRs each. Following a leader sequence, the molecule is composed of the LHR-A closest to the N-terminus comprising a C4b binding domain, by the following two replicates, LHR-B and LHR-C, comprising binding domains for C3b, and by LHR -D antibody closer to the C-terminus followed by two additional SCRs, a putative 25-residue trans-membrane region and a 43-residue cytoplasmic end. CRI is a member of a superfamily characterized by SCR homology. This superfamily contains members which also have a C3 / C4 binding function, such as CR2, C4bp, factor factor. factor Β, and C2, as well as proteins lacking this function, such as receptor for interleukin-2, p2-glycoprotein I, Clr, haptoglobin α chain, and factor XHIb. CRI is known to be a glycoprotein and its deduced amino acid sequence shows 24 potential sites for N-linked oligosaccharides in the extracellular region. However, the synthesis of CRI in the presence of tunicamycin (Lublin et al., J. Biol. Chem. 261: 5736 (1986)) and analysis of the glucosamine content (Sim, Biochem, J. 232: 883 (1985) ) have suggested that only 6-8 of the available sites are in fact bound to oligosaccharides. The N-terminus of the glycoprotein appears to be blocked.

There are four different CRI allotypes that differ in size by 30-50 kD increments. The frequencies of the genes of these allelic polymorphisms (allotypes) differ in the human population (Holer et al., Proc Natl Acad Sci USA 84: 2459-2463 (1987)). The F (or A) alotype is composed of 4 LHR and is about 250 kD; the larger S (or B) alotype contains a LHR fifth which is a chimera of the 5 'half of LHR-B and the 3' half of LHR-A and is expected to have a third binding site for C3b (Wong et al , J. Exp. Med. 169: 847 (1989)), and has about 290 kD. The smaller allotype F '(or C) has increased incidence in patients with systemic lupus erythematosus (SLE) (Van Dyne et al, Clin Exp. Immunol 68: 570 (1987) and Dykman et al., Proc Natl. Acad Sci USA 80: 1698 (1983)) and most likely results in the deletion of LHR-B and a binding site for C3b.

A soluble form of naturally occurring CRI was detected in the plasma of normal individuals and certain individuals with SLE (Yoon & Fearon, J. Immunol., 134: 3332-3338 (1985)). These characteristics are similar to those of the erythrocyte CRI (cell surface) either structurally or functionally.

Hourcade et al (J. Exp. Med. 168: 1255-1270 (1988)) also observed an alternative polyadenylation site in the human CRI transcription unit which was predicted to yield a secreted form of CRI. The mRNA resulting from this truncated sequence comprises the first 8.5 SCRs of the CRI; e.g. the C4b binding domain, and can encode a protein of about 80kD. When a cDNA corresponding to this truncated sequence was transfected into COS cells and expressed, it demonstrated the expected C4b, but not the C3b binding activity (Kyrch et al., F.A.S.E.B J. 3: A368 (1989)). Krych et al. also observed an mRNA similar to that predicted in several cell lines 84 966 ΕΡ Ο 643 608 / ΡΤ 6

and postulated that said truncated soluble form of CRI, which is capable of binding to C4b, can be synthesized in humans.

A number of soluble CRI fragments were also produced by recombinant DNA processes by deletion of the trans-membrane region from the DNAs to be expressed (Fearon et al., International Application No. WO-A-89/09220, published October 5 1989 and Fearon et al., International Application WO-A-91/05047, published April 18, 1991). The soluble CRI fragments were functionally active, since they were able to bind C3b and / or C4b and demonstrated factor I cofactor activity depending on the regions they contained. In addition, they were able to act as inhibitors of in vitro CRI functions, such as oxidative bursting of neutrophils, complement mediated hemolysis, and production of C3a and C5a. A soluble CRI construct, encoded by plasmid sCR1 / pBSCR1c, also demonstrated in vivo activity in a reversed passive Arthus reaction (Fearon et al., 1989 and 1991, Yeh et al., J. Immunol. (1991)) and suppressed inflammation, and post-ischemic myocardial necrosis (Fearon et al., 1989 &1990; Weisman et al., Science 249: 146-151 (1990)). In addition, co-formulation of the sCR1 / pBSCR1c product with the p-anisoylated human plasminogen activator streptokinase (APSAC) complex resulted in an antihemolytic activity similar to that of the isolated APSAC, indicating that the combination of the complement inhibitor, sCR1, with a thrombolytic agent, may be a useful combination therapy (Fearon et al., International Patent Application WOA-91/05047, published April 18, 1991).

Complement receptor-like proteins, which can be purified by the protocol described herein, include allotypes and alleles of CRs, truncated forms, chemically modified forms for example by PEG treatment, and fusion proteins containing a portion of CR. These proteins are designated as being similar to those of the complement receptor, since they possess or retain CR protein properties sufficient to allow purification by the process of this invention. Unless otherwise specifically identified, the term complement receptor protein also includes proteins similar to those of the complement receptor. CR-1-like proteins represent a subset of CR-like proteins including alleles, truncates, chemically modified proteins and fusion proteins derived from the allotype CR-1. The soluble complement 1 receptor (sCR1), defined herein as a soluble form of human CR1 containing all extracellular SCR domains, is a specific example of a CR-1-like protein. 84 966 ΕΡ Ο 643 608 / ΡΤ 7

Complement receptor proteins to be purified according to this invention can be produced by a multiplicity of techniques. If native full length chains are required, then the native molecules can be extracted from the cell sources identified above. When soluble forms are desired, fragments of full-length native molecules are preferred. Accordingly, the DNAs encoding the desired chain fragments are expressed as protein fragments produced by recombination. This invention is particularly useful for the purification of sCR1 from the conditioned cell culture medium of a variety of sCR1 producing recombinant cell lines. While some variation of cell line for cell line and between the various products of the complement receptor may be expected, based on the disclosure contained herein, it is well within the scope of any person skilled in this art to adapt the invention to a particular combination of receptor protein for complement and producer cell line.

Generally, genes encoding proteins such as complement receptors can be cloned by incorporation of the DNA fragments encoding the desired regions of the polypeptide in a recombinant DNA vehicle (eg vector) and transformation or transfection into prokaryotic hosts or eukaryotes. Suitable prokaryotic hosts include, but are not limited to, Escherichia. Streptomvces. Bacillus and the like. Suitable eukaryotic hosts include, but are not limited to, yeasts, such as Saccharomyces. and cultured animal cells such as VERO, HeLa, Cl27 mouse, Chinese hamster ovary (CHO), WI-38, BHK, COS, MDCK. and insect cell lines. Particularly preferred hosts are dihydrofolate reductase-deficient CHO cell lines such as ATCC CRL 1793, CRL 9096 and other cell lines described hereinafter. Said recombinant techniques have now become well known and are described in Methods in Enzvmology. (Academic Press), Volumes 65 and 69 (1979), 100 and 101 (1983), and in the references cited therein. A broad technical discussion embodying the most commonly used recombinant DNA methodologies can be found in Maniatis et al., Molecular Cloning. Cold Spring Harbor Laboratory (1982) or Current Protocols in Molecular Biology. Greene Publishing (1988, 1991).

One way of obtaining a DNA fragment encoding a desired polypeptide such as a complement receptor is by cloning the cDNA. In this process, messenger RNA (mRNA) is isolated from cells recognized or suspected of producing the desired protein. Through a series of enzymatic reactions, the cell mRNA population is copied to a complementary DNA (cDNA). The cDNA 84 966 ΕΡ Ο 643 608 / ΡΤ 8

is then inserted into cloning vehicles and subsequently used to transform a suitable prokaryotic or eukaryotic host. The resulting "cDNA library" is composed of a population of transformed host cells, each of which contains a single gene or a gene fragment. The entire library, in theory, provides a representative sample of the coding information present in the mRNA mixture used as the starting material.

Libraries can be screened using nucleic acid probes or antibody to identify specific DNA sequences. Once isolated, these DNA sequences can be modified or assembled into whole genes. Altematively, as described in this invention, specific fragments of a gene can be engineered independently of the rest of the gene. Protein fragments encoded by these engineered gene fragments may not be found in nature, but may be of significant utility in the treatment of undesirable physiological conditions. Genetically engineering the receptor for the soluble complement for the prevention and / or treatment of changes involving complement activity is one such case.

Once the gene or gene fragment is cloned, the DNA can be introduced into an expression vector and such a construct can be used to transform an appropriate host cell. An expression vector is characterized by having expression control sequences as defined herein, such that when the DNA sequence of interest is functionally linked thereto, the vector is capable of directing the production of the product encoded by the DNA sequence of interest in a host cell containing the vector. With specific reference to this invention, it is possible to assemble fragments of a single coding sequence so that, by expression, a soluble receptor protein is formed. A particularly effective application of this protocol to the production of recombinant SCR1 is found in Fearon et al., PCT applications WO-A-89/09220, published October 5, 1989, and WO-A-91/05047, published on April 1991, cited above.

Once the recombinant product is produced, it is desired to recover the product. If the product is exported by the cell that produces it, the product can be recovered directly from the cell culture medium. If the product is retained intracellularly, the cells must be physically disrupted by mechanical, chemical or biological means in order to obtain the intracellular product.

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In the case of a protein product, the purification protocol should not only provide a protein product which is essentially free of other proteins, which means that it is at least 80% and preferably more than 95% pure relative to the total protein in the preparation , but also eliminate or reduce to acceptable levels other host cell contaminants, DNA, RNA, potential pyrogens and the like.

As mentioned previously, a variety of host cells may be used for the production of the receptors of this invention. The choice of a particular host cell falls well within the scope of the skillfully skilled artisan taking into account, inter alia, the nature of the receptor, its synthesis rate, its rate of degradation and the characteristics of the recombinant vector which directs the receptor expression. The choice of the host cell expression system largely dictates the nature of the cell culture processes to be employed. The selection of a particular mode of production, be it batch or continuous, rotary lifting or air, liquid or immobilized, can be performed once the expression system has been selected. Thus, fluidized bed bio-reactors, hollow fiber bio-reactors, cylindrical vial cultures, or stirred tank bio-reactors, with or without cellular microcarriers, may be variously employed. The criteria for such selection are appreciated in the art of cell culture. They are not detailed herein, since they fall outside the scope of this invention. This invention relates to the purification of receptors for the complement known to exist in a conditioned cell culture medium.

As mentioned above, this invention relates, inter alia, to the application of hydrophobic interaction chromatography (HIC) for the purification and analysis of proteins from the receptor to the complement. Hydrophobic molecules, in an aqueous solvent, will self-associate. This association is due to hydrophobic interactions. It is currently appreciated that macromolecules such as proteins have on their surface extensive hydrophobic plaques in addition to the expected hydrophilic groups. HIC is based, in part, on the interaction of these plates with hydrophobic ligands attached to chromatographic supports. A hydrophobic ligand coupled to a matrix is variously designated herein as a carrier for HIC, HIC gel or HIC column. It is further appreciated that the strength of the interaction between the protein and the support for HIC is not only a function of the proportion of non-polar surfaces relative to the polar ones in the protein, but also by the distribution of the non-polar surfaces.

A number of matrices may be employed in the preparation of HIC columns, the most widely used being agarose. Silica and organic polymer resins may be used. Useful hydrophobic ligands include, but are not limited to, alkyl groups having from about 2 to about 10 carbon atoms, such as butyl, propyl or octyl; or aryl groups such as phenyl. Conventional HIC products for gels and columns can be obtained commercially from suppliers such as Pharmacia LKB AB, Uppsala, Sweden, under the names of butyl-SEPHAROSE®, phenyl-SEPHAROSE® CL-4B, octyl-SEPHAROSE® FF and phenyl-SEPHAROSE® FF; Tosoh Corporation, Tokyo, Japan under the product names TOYOPEARL Butyl 650M (Fractogel TSK Butvl-650) or TSK-GEL Phenyl-5PW; Miles-Yeda, Rehovot, Israel, under the name alkyl-agarose product, wherein the alkyl group contains 2-10 carbon atoms, and JT Baker, Phillipsburg, NJ, under the product name Bakerbond WP-HI-propyl .

It is also possible to prepare the desired HIC column using standard chemistry. For example, matrix / ligand combinations of the form NH, i '

Wherein M is a matrix such as agarose, may be formed upon activation by the cyanogen bromide of the agarose by coupling with an alkylamine as taught by Er- , et al., Biochem Biophys Res, Comm 49: 383 (1972) Alternatively, combinations of the form

Wherein M is a matrix such as agarose and A is aryl, may be prepared by a compound of the formula wherein M is a matrix such as agarose and A is aryl; Glycidyl ether coupling process (Ulbrich, V. et al., Coll., Czech Chem., Comm., 9: 1466 (1964)). Briefly, a gel, generally agarose, is transferred to an organic solvent, e.g. dioxane. This is done stepwise (100 ml portions to 100 ml sedimented gel) on a Btichner funnel: (1) a water-dioxane (4: 1) wash, (2) a water-dioxane wash (3: 2), (3) a wash with water-dioxane (2: 3), (4) a wash with water-dioxane (1: 4) and (5) seven washes with dioxane. 100 ml of dioxane are added to 100 ml of sedimented gel and 2 ml of a 48% solution of boron trifluoride etherate in diethyl ether are added and stirred for five minutes. 1 ml of the appropriate glycidyl ether dissolved in 10 ml of dioxane was added dropwise from a separatory funnel. The reaction takes about 40 minutes. At the end of the reaction, the derivative gel is transferred back to an aqueous medium but reversing steps (4) through (1)

84 966 ΕΡ Ο 643 608 / ΡΤ 11 and ending with a final wash in water. The amount of ligand to be coupled to the gel can be controlled by varying the amount of glycidyl ether added to the reaction mixture. The reaction may be represented generally as follows:

The OH /? BF, Et;

Wherein M is a matrix such as agarose and R is alkyl or aryl. The density of the ligand is an important parameter, since it influences not only the strength of the interaction but also the capacity of the column. The ligand density of the commercially available phenyl or octylphenyl gels is in the range of 40 Limoles / ml of gel bed. The capacity of the gel is a function of the particular protein in question, as well as the pH, temperature and saline concentration, but in general it can be expected to be in the range of 3-20 mg / ml gel. The choice of a particular gel may be determined by the person skilled in the art. In general, the strength of the interaction of the HIC protein and ligand increases with the chain length of the alkyl ligands, but ligands having 4 to about 8 carbon atoms are suitable for most of the separations. A phenyl group exhibits approximately the same hydrophobicity as a pentyl group, although the selectivity may be quite different by virtue of the possibility of the interaction. pi-pI with aromatic groups on the protein. Adsorption of the proteins to an HIC column is favored by high salt concentrations, but actual concentrations may vary over a wide range depending on the nature of the protein and the particular HIC ligand chosen. Several ions may be ordered in a series called soluphobic, depending on whether they promote hydrophobic interactions (desalting effects) or break the water structure (chaotropic effect) and lead to the weakening of the hydrophobic interaction. The cations are ordered in terms of increasing desalting effect such as Ba &quot; &lt; Ca &quot; * &lt; Mg &quot; * &lt; Li &quot; &lt; Cs &quot; &lt; In &quot; &lt; K &quot; &lt; Rb &quot; &lt; NH4 &quot;. However, anions can be ordered in terms of increasing chaotropic effect such as PO / &quot; &lt; S04 &quot; &lt; CH3COO '&lt; Cf < Br < NO./ &lt; CIO / < Γ < SCN '.

Thus, salts may be formulated which influence the strength of the interaction as determined by the following ratio:

84 966 ΕΡ Ο 643 608 / ΡΤ 12

Na, SO4 &gt; NaCl &gt; (NH4), SO4 &gt; NH4 Cl &gt; NaBr &gt; NaSCN

In general, saline concentrations of ammonium sulphate of between about 0.75 to about 2 M or of NaCl between about 1 and 4 M. are useful. The influence of temperature on the separations of the ICC is not simple, although generally a reduction decrease the interaction. However, any benefit that could arise from the increase in temperature should also be weighed against adverse effects such an increase may have on the activity of the protein. The elution, either stepwise or in the form of a gradient, may be obtained in several ways: (a) by modifying the salt concentration, (b) by changing the polarity of the solvent or (c) by adding detergents. By decreasing the salt concentration the adsorbed proteins are eluted in the order of increasing hydrophobicity. Changes in polarity may be affected by additions of solvents such as ethylene glycol or (iso) propanol, thereby reducing the strength of the hydrophobic interactions. Detergents function as protein shifters and were used primarily in connection with the purification of membrane proteins.

As mentioned above, HIC is particularly useful when used in combination with other techniques for the purification of proteins. This means that the application of HIC to material which has been partially purified by other processes for the purification of proteins is preferred. By the term "partially purified" is meant a protein preparation in which the protein of interest is present in at least 5 weight percent, more preferably at least 10%, and most preferably at least 45%. Accordingly, the application of HIC is best appreciated in the context of a global purification protocol for complement receptor proteins. It has been found useful, for example, to submit a sample of the conditioned cell culture medium to partial purification prior to the application of the HIC. By the term &quot; conditioned cell culture medium &quot; is meant a cell culture medium which sustained cell growth and / or cell maintenance and contains secreted product. A concentrated sample of said medium is subjected to one or more steps for purification of the proteins prior to the application of an HIC step. According to the present invention, the sample is first subjected to cation exchange chromatography before the IIC is performed. As mentioned above, various anionic or cationic substituents may be attached to dies in order to form anionic or cationic carriers for chromatography. Substitution

Anion exchange includes diethylaminoethyl (DEAE), quaternary aminoethyl (QAE) and quaternary amine groups (Q). Cation exchange substituents include carboxymethyl (CM), sulfoethyl (SE), sulfopropyl (SP), phosphate (P) and sulfonate (S). Ion-exchange cellulosic resins such as DE23, DE32, DE52, CM-23, CM-32 and CM-52 are available from Whatman Ltd. Maidstone, Kent, United Kingdom. SEPHADEX® crosslinked ion exchangers are also known. For example, DEAE-, QAE-, CM-, and SP-SEPHADEX® and DEAE-, Q-, CM-, and S-SEPHAROSE® are all available from Pharmacia AB. Further, both the DEAE and CM derivatives of the ethylene glycol-methacrylate copolymer, such as TOYOPEARL DEAE-650S and TOYOPEARL CM-650S, are available from Toso Haas Co., Philadelphia, Pa. Since elution from ionic supports generally involves the addition of salt and since, as mentioned previously, the HIC is intensified under increased salt concentrations, the present invention comprises an HIC step following a cation exchange chromatographic step, and optionally after a further purification step mediated by salt. It is preferred that the cation exchange chromatographic step and the precipitation step of ammonium sulfate precede the application of the HIC. According to the present invention, a size exclusion chromatography step is carried out after the HIC step. Additional purification protocols may be added, including but not necessarily limited to additional ion exchange chromatography, size exclusion chromatography, viral inactivation, concentration and freeze-drying.

When the resulting HIC eluate is subjected to additional ion exchange chromatography, it is preferred that both anionic and cationic processes are employed.

As mentioned above, gel filtration chromatography affects the separation based on the size of the molecules. It is actually a form of molecular sieving. It is desirable that there is no interaction between the matrix and the solute, and therefore fully inert matrix materials are preferred. It is also desirable that the matrix be rigid and highly porous. For large-scale processes, stiffness is the most important since that parameter establishes the overall flow rate. Traditional materials, e.g. SEPHADEX® or BIO-GEL®, have been shown to be sufficiently inert and available in a range of pore sizes. however these gels have been shown to be relatively soft and not particularly well suited for large-scale purification. More recently, gels of increased stiffness (e.g., SEPHACRYL®, UTROGEL®, FRACTOGEL® and SUPEROSE®) have been developed. All of these materials are accessible in particle sizes that are smaller than those

84 966 ΕΡ Ο 643 608 / ΡΤ 14 available on traditional media, so the resolution is maintained even at higher flow rates. Auxiliary dies of series TOYOPEARL HW (Toso Haas) are preferred.

For purposes of illustration only, this invention has been applied to the purification of a receptor for the complement of the soluble type. More specifically, to a soluble CRI construct containing control regions, LHR-A, LHR-B, LHR-C, LHR-D, SCR29, SCR30 up to and including the first trans-membrane region alanine residue; and corresponding to the sequences encoding CR1 in the plasmid pBSCR1 of Fearon et al., 1989, International Patent Application No. WO-A-89/09220, published October 5, 1989 (hereinafter "TP10HD"). Construction of a recombinant system for the production of TP10HD is detailed in the above-mentioned PCT application and summarized as follows. CHO cells were trypsinized and seeded at 5 x IFC per 60 mm dish and left in the growth medium (Hams F12 nutrient medium (041-1765) with 1% glutamine broth (043-05030), pen / strep broth 1% (043-05070) and 10% fetal calf bovine serum (011-6290), Gibco, Paisley, Scotland) at 37øC in a humidified incubator under an atmosphere of 5% CO: 95% air . After 21 hours the cells were used for transfection of the DNA. An expression plasmid containing the sCR1 coding sequence of pBSCR1c was cotransfected with pSV2dhfr into a Chinese Hamster Ovary cell line requiring dhfr (CHODUXBII). The transfection was performed in growth medium and employed the calcium / coprecipitation process for glycerol shock as described in DNA Cloning, D. M. Glover ed. (Chapter 15, C. Gorman). After transfection with pBSCR1 / pTCSgpt and pSV2dhfr, the cells were maintained in growth medium for 46 hours under growth conditions (as described above) prior to the selection procedure. The selection and co-amplification process was carried out essentially as described by R. J. Kaufman et al. (Mol. Cell Biol. 5: 1750-1759 (1985)). Forty-six hours post-transfection the cells were changed to MEM ALPHA (041-02571), 1% glutamine broth, 1% pen / strep broth (043-05070) and dialyzed fetal calf bovine serum ( 220-6300AJ) (Gibco, Paisley, Scotland). Cells were maintained in the selective medium for 8-10 days until dhfr 'colonies appeared. When colonies were established the cells were switched to a methotrexate-containing selective medium (A6770, Sigma Chem Co., St. Louis, Mo.). The concentration in methotrexate was initially 0.02 μΜ and increased step by step up to 5 μΜ. During the amplification process, aliquots of growth medium from the growing cells were analyzed for 84 966 ΕΡ Ο 643 608 / ΡΤ 15

production of TP10HD by ELISA. Any recombinant complement receptor secretory cell line (e.g., ATCC CRL 10 052) may be used to provide the conditioned medium for purification according to this invention, but a particular cell line is certainly not required.

A transfected CHO cell line capable of producing TP10HD can be cultured by a variety of cell culture techniques. For the application of this invention the particular method of culturing is not critical, however for purposes of illustration, a method for culturing cells which may be used is a continuous perfusion process based on the Verax fluidized bed technology as embodied in US-A-4,861,714; US-A-4 863 856; US-A-4 978 616 and US-A-4 997 753. Thus, transfected cells such as those described above are staggered in CCM-3 medium (a mixture of DMEM, Ham F-12, bovine serum albumin and the like supplements) supplemented with 10% fetal bovine serum (FBS) and 5 mM methotrexate (MTX). The cell population was expanded in cylindrical vials until a sufficient number of cells were available for inoculation of a bioreactor. Prior to inoculation, an S200 bioreactor was subjected to On-site Clean-Up Cycles (CIP) and On-Site Vaporization (SIP) cycles. It was then filled with CCM-3 medium containing 5% FBS and loaded with 450 grams of microspheres. Microspheres were conditioned with medium prior to inoculation. The reactor was inoculated with cells and the operating parameters were: pH 7.2, 37øC, 02 dissolved in the inlet (fluidized bed bottom) between 100 and 400 torr (1 torr = 133 Pa), O, dissolved in the outlet (top of the fluidized bed) between 0 and 200 torr. Following an initial batch phase, the medium was perfused, with periodic rate increases to maintain the glucose concentration at 1.0 g / l. This continued until a sufficient number of cells had accumulated in the reactor to inoculate an S2000 bioreactor. After CIP and SIP, an S-2000 reactor was filled with CCM-3 medium supplemented with 5% FBS and 5 mM MTX and loaded with 5000 grams of microspheres. These microspheres were conditioned with medium prior to inoculation. The operating conditions for the temperature, reactor arrangement and dissolved O are as given above. The microspheres of the S-200 reactor were aseptically transferred to the S-2000 reactor in order to initiate the batch phase. When the glucose concentration fell below 1.5 g / l, the growth phase was triggered by initiation of medium infusion (CCM-3, 5% FBS and 5 mM MTX) at a rate sufficient to maintain the concentration of glucose in 1.0 g / l. Cell growth was monitored in-line by measuring the rates of oxygen uptake and glucose uptake. When sufficient cells accumulated inside the reactor, the perfusion medium was changed to CCM-3 transition medium supplemented with 1% FBS and 5 mM MTX. Again, this infusion rate was modified to

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Maintaining a glucose concentration of 1.0 g / l. Upon further growth in the transition medium, the perfusion medium was once again exchanged into the production medium, CCM-3 supplemented with 5 mM MTX. The perfusion rate was increased in order to maintain a glucose concentration of LO g / 1. Thereafter, the set points of either the O, dissolved at the outlet or the recirculation flow rate were reduced in order to maintain control over the reactor. The production phase typically lasts for about 60 days.

Between 400 and 1600 liters of reactor permeate, stored at 4-8 ° C, were processed through a Millipore Prostak Microfiltration Unit. The cell-free permeate from this operation supplied the ultrafiltration step. The permeate was concentrated 30-60 X with a Millipore Spiral Winding System. After concentration, the retentate was drained to a holding tank and the system was filled with 5-20 l of 50 mM phosphate buffer, pH 7.5. The wash buffer was drained from the system and combined with the retention. The ultrafiltration concentrate was filtered through a prefilter and a 0.22 mm end filter into a pre-autoclaved Nalgene flask. Nominally, 800 ml of concentrate is dispersed into each vial and stored under freezing.

As mentioned above, the particular recombinant production system and the particular cell culture protocol are outside the scope of this invention. The system and protocol discussed above are representative of the many options available to the person skilled in the art and are only included herein for purposes of illustration. The purification protocol which is the aim of this invention is applicable, with routine modifications only, to a variety of receptor proteins for recombinant and receptor-like complement regardless of how they are produced or cultured.

The purified complement receptor proteins obtained by practicing the process of this invention have the following properties: 1) more than 95% by weight CR protein; 2) stability to proteolytic degradation at 4 ° C for at least three months; 3) low endotoxin (<1 U./mg protein); 4) low DNA (<1pg / mg protein); 5) non-CR protein &lt; 5% by weight; and 6) virally inactive. The following examples further illustrate this invention, but are not presented with the intention of limiting the appended claims.

84 966 ΕΡ Ο 643 608 / ΡΤ 17

Example I Introduction The process outlined below was developed for the isolation and purification of the receptor for soluble complement 1 (sCR1) from conditioned cell culture medium concentrate. This process is designed to prepare sCR1 of protein purity> 95%, while impurities derived from the host cell, cell culture medium, or other raw materials are removed. The recovery process is composed of nine steps including cationic and anion exchange chromatography, hydrophobic interaction, and size exclusion; a precipitation in ammonium sulphate; and two viral inactivation treatments. Each step is described in detail below. Steps 1 through 5 are carried out at 2-8 ° C, and steps 6 through 9 are carried out at 20-25 ° C.

Step 1: Pretreatment of medium

Under stirring, conditioned medium concentrate is adjusted to pH 5.2 by the addition of 1N HCl. When the addition is complete, stirring is continued and the pH monitored for 10 min. Adjusting the pH produces a heavy precipitate. Clarification is obtained by microfiltration through a series of 3 series-linked Millipore Polygard-CR filters (5 pm to 0.5 pm to 0.1 pm). The sCR1 is recovered in the filtrate. Acidification and filtration of the medium concentrate removes both non-sCR1 protein and non-proteinaceous material; and adjust the sCR1-containing filtrate to the appropriate pH for the subsequent S-SEPHAROSE chromatography.

Step 2: Rapid flow chromatography on Pharmacia S-SEPHAROSE The filtrate at pH 5.2 is charged to a Pharmacia S-SEPHAJROSE Rapid Flow gel column previously equilibrated with Buffer A at a flow rate of 60 cm / h, and a capacity of <3 grams of bed volume sCRl /.. The column is washed at 150 cm / hr with 3 to 5 volumes of Buffer A bed, followed by 5 to 10 Buffer B bed volumes. SCR1 binds to the column and is eluted with Buffer C. The column is stripped by washing with 3 volumes of the Buffer D bed.

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S-SEPHAROSE chromatography removes a large proportion of impurities derived from cells and medium (particularly proteins) and concentrates sCR1 in Buffer C eluted from the column for further processing.

Step 3: Ammonium sulphate precipitation C-Buffer C eluted from S-SEPHAROSE is adjusted to 1.2 M ammonium sulfate by the addition of Buffer E. When the addition is complete, stirring is stopped, and the mixture is allowed to stand all-night. The sCR1-containing precipitate is collected by centrifugation at 8000 x G for 10 min. The pellet material is resuspended by gentle shaking in approximately 4 L of Buffer F. Additional Buffer F is added until the absorbance of the solution at 280 nm is 1.5 units of O.D. The solution is stirred overnight, and filtered through a 0.1 μm Millipore Polygard-CR filter. Precipitation on ammonium sulfate removes additional impurities, and prepares sCR1 for chromatography by hydrophobic interaction.

Step 4: TOYOPEARL Butvl-650M Chromatography The re-solubilized and filtered ammonium sulfate pellet is adjusted to 0.8 M ammonium sulfate by the addition of Buffer E under stirring.

When the addition is complete, the mixture is loaded onto a TOYOPEARL Butyl-650 M gel column previously equilibrated with Buffer G at a flow rate of 150 cm / hr, and a capacity of &lt; 4 grams of total protein / 1 column volume. Buffers and column are maintained at 2-8 ° C. When loading is complete, the column is washed with 2-3 volumes of the Buffer G bed, washed with 3 volumes of the Buffer H bed, and the bound sCR1 eluted with Buffer I.

Solid GuHCl is added to the Butil-Buffer I eluate to a concentration of approximately 2M and stirred until completely dissolved. The pH is monitored and if necessary adjusted to pH 7.0 using 2.5 N NaOH.

When the GuHCl is completely dissolved, the solution is stored for 6 minutes and charged to a Pharmacia SEPHADEX G25 column previously equilibrated with Buffer J at a flow rate of 60 cm / hr. The volume of the load should not exceed 25% of the volume of the SEPHADEX G25 column.

After the sCR1 is eluted (elutes in void volume), the column is washed with Buffer J until the "salt" peak has eluted and the conductivity has returned to the baseline level. The product of SEPHADEX G25 is adjusted to pH 11 by the addition of 2.5 M NaOH, the solution is maintained at pH 11 for 16 minutes, and readjusted to pH 9.0 using 2.5 HC HCl. The material is now ready for anion exchange chromatography.

Treatments with GuHCl and pH 11 provide retroviral inactivation if any virus is present and chromatography on SEPHADEX G25 prepares the sCR1 product for DEAE TOYOPEARL chromatography.

Step 6: TOYOPEARL DEAE-650S Chromatography The SEPHADEX G25 product is loaded onto a TOYOPEARL DEAE-650S gel column, previously equilibrated with Buffer J, at a flow rate of 150 cm / hr and a capacity of &lt; 6 grams of protein / 1 column volume. After loading, the column is washed with 3 volumes of Buffer J. The bound sCR1 is eluted with a linear gradient of 5 column volumes starting from 100% Buffer J and extending to 100% Buffer K. column is stripped by lavage with 3 volumes of Buffer L. Toxoplasma DEAE chromatography removes contaminating proteins, DNA, and potential viral impurities.

84 966 ΕΡ Ο 643 608 / ΡΤ 20

Step 7: Chromatography on TOYOPEARL CM-650S

Dilute the DEAE product with 2 volumes of Buffer M and adjust to pH 5.5 with 2.5 N HCI. Charge the diluted mixture to a column of TOYOPEARL CM-650S gel, previously equilibrated with Buffer M, at a rate of flow rate of 150 cm / hr, and a capacity of &lt; 11 grams of protein / 1 column volume. After loading is complete, 3-bed lavage of the Buffer M column and elution of the sCR1 bound with a linear gradient of 5 column volumes, ranging from 100% Buffer M to 100% Buffer N. The column is stripped with 3 volumes of the Buffer O-bed. The eluate containing the product is neutralized with 1/10 volumes of 0.5 M dibasic sodium phosphate, and is now ready for size exclusion chromatography. Chromatography in TOYOPEARL CM removes contaminating proteins, DNA, and potential viral impurities.

Step 8: TOYOPEARL HW65S Chromatography The TOYOPEARL CM product is loaded onto a TOYOPEARL HW65S column, previously equilibrated with Buffer F, at a flow rate of 30 cm / hr. The load volume shall not exceed 5% of the total TOYOPEARL HW65S column volume. Collect the peak of the complete product until the absorbance decreases up to 10% of the maximum absorbance. The material is now ready for the final concentration. Size exclusion chromatography removes the last traces of impurities from low molecular weight proteins, and serves to exchange the sCR1 of buffer to the final target buffer.

Step 9: Concentration and final filtration The TOYOPEARL HW65S combination is concentrated to 5-6 mg / ml using a Pharmacia Minisette ultrafiltration unit adapted with an Omega 100 K MWCO membrane. The concentrated product is filtered through a Millipak Millipak 0.2 μm filter. 84 966 ΕΡ Ο 643 608 / ΡΤ 21

Buffer Caps Buffer Buffer Buffer Buffer Buffer Buffer Buffer Buffer Buffer Buffer Buffer Buffer Buffer Buffer Buffer Buffer Buffer Buffer Buffer Buffer Buffer Buffer Buffer Buffer Buffer Buffer Buffer Buffer Buffer sodium phosphate, 100 mM NaCl, pH 6.0 20 mM sodium phosphate, 500 mM NaCl, pH 7.0 1 M NaCl 3M ammonium sulfate, 100 mM sodium phosphate, pH 7, 10 mM sodium phosphate, 0.9% w / v NaCl, 0.8 M pH 7.8 ammonium sulfate, 100 mM sodium phosphate, pH 7.0, 0.7 M ammonium sulfate, 100 mM sodium phosphate, pH 7.0 0.09 M ammonium sulfate, 100 mM sodium phosphate, pH 7.0 50 mM Tris / Tris.HCl, pH 9.0 50 mM Tris / Tris.HCl, 0.2 M NaCl, pH 9.0 50 mM Tris / Tris.HCl, 1.0 M NaCl, pH 9.0 50 mM MES / MES.Na, pH 5.5 50 mM MES / MES .At. 0.25 M NaCl, pH 5.5 50 mM MES / MES.Na, 1.0 M NaCl, pH 5.5 (Table below)

23 # 84 966 ΕΡ Ο 643 608 / ΡΤ

Lisboa, 16. ΛδΟ. 2300

By AVANT IMMUNOTHERAPEUTICS, INC.

Claims (12)

A method for purifying a protein from the receptor for the complement or a fragment thereof, a protein similar to the complement receptor or a fragment thereof from a mixture containing the (a) contacting the mixture with a chromatographic carrier for cation exchange and selective elution of the protein, (b) contacting the protein eluted from step (a) with a chromatographic carrier for hydrophobic interaction and selective elution of the protein, (c) contacting the protein eluted from step (b) with a size exclusion chromatographic support and selective elution of the protein. and optionally also comprising any one or more of the following steps: precipitation on ammonium sulfate prior to chromatography by hydrophobic interaction, additional ion exchange chromatography, size exclusion chromatography, viral inactivation, concentration, and freeze-drying. A method according to claim 1, wherein the protein is CRI and / or fragments thereof. A method according to Claim 1 or Claim 2, wherein: (a) the cation exchange chromatographic support is selected from the group consisting of carboxymethyl substituted cellulose, carboxymethyl substituted crosslinked dextrans, crosslinked sulphopropyl dextrans , carboxymethyl substituted agarose beads, sulfonate substituted agarose beads, carboxymethyl substituted ethylene glycol-methacrylate copolymers and elution is by addition of buffered saline; (b) the chromatographic carrier for hydrophobic interaction is selected from the group consisting of C 2 -C 8 -alkyl agarose, aryl agarose, alkyl silica, organic alkyl polymer resin; and 84 966 ΕΡ Ο 643 608 / ΡΤ 2/6 (c) Chromatographic carrier for size exclusion is selected from crosslinked polyacrylamides and ethylene glycol-methacrylate copolymers. A method according to Claim 3, wherein: (a) the cation exchange chromatographic support is sulfonate substituted agarose beads and the buffered salt is 20 mM sodium phosphate, 500 mM NaCl, pH 7.0 ; (b) the chromatographic carrier for hydrophobic interaction is a butylethylene glycol-methacrylate copolymer and the protein is selectively eluted with a buffer of 100 mM sodium phosphate, pH 7.0 containing 0.09 M ammonium sulfate; and (c) the size exclusion chromatographic support is a copolymer of ethylene glycol-methacrylate and the elution is with 10 mM sodium phosphate, 0.9% w / v NaCl, pH 7. The method according to Claim 1 or Claim 2, wherein a complement receptor protein of type 1 or a fragment thereof is purified from conditioned cell culture medium containing the same comprising the steps of: ( (a) subjecting the culture medium to a first cation exchange chromatography, (b) subjecting the product of step (a) to precipitation in ammonium sulfate, (c) subjecting the product of step (b) to hydrophobic interaction chromatography, (d) subjecting the product of step (c) to anion exchange chromatography, (e) subjecting the product of step (d) to a second cation exchange chromatography, and (f) subjecting the product of step (e) to chromatography of exclusion by size. A method according to claim 5, wherein: 84 966 ΕΡ Ο 643 608 / ΡΤ 3/6 (a) the first cation exchange chromatography employs a support selected from the group consisting of carboxymethyl substituted cellulose, carboxymethyl substituted crosslinked dextrans, sulfopropyl substituted crosslinked dextrans, carboxymethyl substituted agarose beads, sulfonate substituted agarose beads, carboxymethyl substituted ethylene glycol-methacrylate copolymers and the elution is by a buffered saline solution; (b) the ammonium sulfate is present in a concentration of 1.2 M; (c) the chromatographic support for hydrophobic interaction is selected from the group consisting of C 2 -C 8 -alkyl agarose, aryl agarose, alkyl silica, organic alkyl polymer resin; (d) anion exchange chromatography employs a carrier selected from the group consisting of diethylaminoethyl substituted cellulose, diethylaminoethyl substituted or quaternary aminoethyl substituted crosslinked dextrans, diethylaminoethyl substituted or quaternary amine substituted aromatic beads, and ethylene glycol copolymers diethylaminoethyl substituted methacrylate; (e) the second cation exchange chromatography employs a carrier of carboxymethyl substituted ethylene glycol-methacrylate copolymers; (f) size exclusion chromatography employs a carrier selected from crosslinked polyacrylamides and ethylene glycol-methacrylate copolymers. A method according to Claim 6, wherein: (a) the support for the first cation exchange is sulfonate-substituted agarose beads and the buffered saline is 20 mM sodium phosphate, 500 mM NaCl, pH 7 , 0; (b) the chromatographic carrier for hydrophobic interaction is a butylethylene glycol-methacrylate copolymer and the protein is selectively eluted with a buffer containing 100 mM sodium phosphate, pH 7.0 containing 0.09 mM ammonium sulfate; 84 966 ΕΡ Ο 643 608 / ΡΤ 4/6 (c) The support for the anion exchange employs a ethylene glycol-methacrylate copolymer substituted with diethylaminoethyl; and (d) the carrier for size exclusion is a copolymer of ethylene glycol-methacrylate. A method according to Claim 1, wherein a complement receptor protein or fragment thereof, a complement receptor-like protein or a fragment thereof, is purified from a conditioned cell culture medium, comprising: (a) concentration of the conditioned cell culture medium; (b) adsorbing the protein from the receptor to the complement on a chromatographic support for cation exchange; (c) washing the adsorbed protein with at least one buffer; (d) elution of the washed protein; (e) precipitation of the protein with ammonium sulfate; (f) re-solubilizing the precipitated protein; (g) adsorbing the solubilized protein from step (f) onto a chromatographic carrier for hydrophobic interaction; (h) selective elution of the protein; (i) adsorbing the eluate from step (h) onto an anion exchange support; (j) eluting the adsorbed protein; 84 966 ΕΡ Ο 643 608 / ΡΤ 5/6 (k) adsorption of the eluate from step (j) onto a support for cation exchange; (l) elution of the adsorbed protein; (m) submission of the eluate from step (1) to size exclusion chromatography; and (n) recovering the protein therefrom. A method according to any one of Claims 1 to 8, which comprises an optional step following hydrophobic interaction inactivation chromatography of virus. A method according to claim 8, wherein: the cation exchange support of step (b) is sulfonate substituted agarose beads; the eluate from step (d) is adjusted to 1.2 M ammonium sulfate; the cation exchange support of step (k) is a carboxymethyl substituted ethylene glycol-methacrylate copolymer; the anion exchange carrier is a diethyleneaminoethyl substituted ethylene glycol-methacrylate copolymer; the chromatographic carrier for hydrophobic interaction is a copolymer of butylethylene glycol-methacrylate; and size exclusion chromatography employs an ethylene glycol-methacrylate copolymer. A method according to claim 8, wherein said recovery step (n) is performed by combining and concentrating the protein containing fractions from the final chromatography step (m) by ultrafiltration. 84 966 ΕΡ Ο 643 608 / ΡΤ 6/6 A method according to Claim 8 which comprises the step following step (h) elution of hydrophobic interaction inactivation of virus, if present, by treatment of the eluate from the chromatography by hydrophobic interaction with base and with hydrochloride of guanidine. Lisbon, &lt; 6. m 2800 By AVANT IMMUNOTHERAPEUTICS, INC. - THE OFI AGENT EMOTION OF THE CUMHA FERREIRA DEPARTMENT OF AGRICULTURE, INC. 1600 LISBON